笔者想感谢资助号UO1CA143069，R01CA142833，R01CA114462，RO1CA179556，T32-AG000213-24和T32-GM008692-18资助这项工作。我们也承认位点杰里米Bredfelt和玉明刘为发展和协助与CT-FIRE分析。

1.中和，稀释和3D调查和细胞收缩测定胶原蛋白溶液聚合<ol><li>在无菌组织培养罩冰，中和胶原（1:1）用无菌，冰冷的100mM HEPES在2×PBS中，pH为7.4，在15ml锥形管中。用塑料吸管调匀直至溶液均匀混合漩涡不再可见。要小心，不要在混合过程中引入气泡。简单地存放在冰上。 </li><li>稀释中和胶原蛋白与细胞介质适当浓度（如RPMI，DMEM 等 ）。 <ol><li>来计算，以弥补所需胶原浓度所需中和胶原蛋白的量，使用等式N =（D * V）/（S / 2），其中N为所需中和胶原蛋白的量，D是所需的胶原浓度，V是所需胶原浓度的最终体积，以及S是叔他首发股票的胶原蛋白浓度。 </li><li>通过加入细胞和细胞培养基溶液的中和胶原的量带来补足体积。调匀并在冰上储存直至准备施法。 <ol><li>例如:对于在1ml凝胶体积有2毫克/毫升凝胶（典型体积为在6孔板或50毫米的玻璃底皿的凝胶浇铸）中，首先通过将所需最终体积乘以所希望的浓度（2毫克/毫升） （1毫升）中。借此号码（2毫克）和由瓶列出的原料浓度（9.49毫克/毫升）的一半除以它。在这种情况下，0.42毫升中和胶原蛋白与0.58毫升介质/细胞混合物的稀释。 </li></ol></li></ol></li><li>吸管冰冷胶原/细胞/培养基混合物变成一个非组织培养物处理的6孔板或50毫米的玻璃底培养皿中。用枪头均匀摊开的解决方案。 注意:使用非组织培养处理板向细胞的附着或生长减少collag之外是很重要恩凝胶。 </li><li>允许在室温下聚合大约10 - 15分钟。凝胶应当在聚合变成不透明的。 </li><li>它已经变成不透明后，移动板或盘，以37℃额外45 - 60分钟以完成聚合。 </li><li>经过45 - 通过运行以及周围或盘周边P200的枪头从井两侧的3ml媒体和释放凝胶 - 60分钟，加2。漩涡菜轻轻地释放凝胶。胶原凝胶应该在媒体上浮动。 </li></ol> 2.免疫印迹，细胞形态和凝胶含量Gontractility <ol><li>评估与蛋白质水平，形态或细胞收缩到ECM刚度，浇胶原凝胶开始，按照1.1 - 1.6，与细胞的接种7 - 10天检测。根据增长率和融合凭经验确定每个细胞系播种量。为10天的试验中，接种密度的范围可以从20,000 - 100,000个细胞/凝胶。 </li><li>测量细胞收缩，通过使用尺子或照相机每24小时或在适当的时间间隔测量的凝胶直径。 注意:另外，从显微镜收集对应的图像可以检查在凝胶接种，如腺泡状结构，上皮小管，蜂窝突起，和lameliapodia细胞系的形态特征的特征。 </li><li>为了评估蛋白质水平，如在沃兹尼亚克等人 17和Gallagher 等人 18所述裂解在RIPA凝胶缓冲液和处理为感兴趣的蛋白质的Western印迹分析。 <ol><li>重要信息:在细胞系之间的收缩的比较，正常化收缩到总DNA，其可从凝胶中提取由吕所概述， 等人 19，或到一个不变，管家蛋白（组蛋白，GAPDH 等 ）通过Western。印迹分析，如在沃兹尼亚克等人 17和Gallagher 等人所述。18。如果凝胶内的细胞计数使用血球，确保正常化细胞数占总凝胶面积承包凝胶将集中细胞。 </li></ol></li><li> 4天通过除去将1ml培养基并用1ml新鲜培养基更换它 - 每3饲料凝胶。确保测量之后的加入新鲜的媒体/血清会引起收缩穗养活凝胶。 </li></ol>胶原蛋白光纤对准PDMS微通道3代注:要对齐产生胶原基质，用于PDMS微通道（ 图 2A）的模具，需要通过软光刻15取得了SU-8硅主。 <ol><li>为了使PDMS通道，彻底的一次性​​杯具一门手艺棒混合PDMS。对于一个6英寸的硅主人，用2克固化剂混用20克橡胶基地。 </li><li>解气体的PDMS混合物通过的真空压力下放置一次性杯在真空室550毫米汞柱。解气体为1 - 1.5小时。 </li><li>虽然脱气的PDMS，准备硅主浇注。通过将透明胶片丢球到烤盘其次是有机硅主做准备。确保微模具朝上。 注:在PDMS浇铸和固化，硅氧烷主将透明膜的两片之间夹入。 </li><li>后1 - 1.5小时，从真空室取出脱气的PDMS，慢慢倒在硅氧烷主。 重要提示:避免气泡。继续主中心纷至沓来，让重力均匀地传播它。 注:PDMS下降并不需要一直延伸到主的边缘。 </li><li> PDMS已经被倾倒在主后，对硅主的PDMS上应用投影胶片的第二片。小心地倒在PDMS /硅胶高手顶部滚动第二透明薄片，以避免产生气泡。不着急。 PDMS应均匀地散布现在Ø版本高手。 </li><li>轻轻地放在透明胶片上的橡胶片，随后通过1/8"丙烯酸系片材。 </li><li>在压克力板顶部添加3个10磅的重量。最初，权重将"浮动"。让他们进一步处理之前解决和稳定。 </li><li>设置加热板的温度至70℃，固化的PDMS 4小时。允许干扰前冷却1小时的额外最低。 </li><li>小心地从晶片剥离透明膜的顶片，并用钳子取出频道。 </li><li>存放在无尘的菜，直到准备使用。 </li></ol> 4. PDMS预学习的微通道使用<ol><li>地方频道上新的，干净的透明胶片倒挂。清洁所有端 ​​口（ 图2b），使用打圈用锋利的钳子。删除PDMS中的任何片段。 </li><li>清洁通道使用了一块打包带作为钉布。将胶带粘工作台表面（粘面朝上），然后设置通道0■首页。向下按压钳的圆端渠道，以确保良好的接触。删除，直到可见的碎屑被去除两侧重复。 </li><li>转移清洗，坦然PDMS渠道到50毫升锥形用70％乙醇。在涡旋30秒的最大速度。丢弃EtOH中并用新鲜的70％乙醇取代。储存在70％乙醇中待用。 </li><li>在组织培养罩，并使用无菌技术，传输PDMS渠道，一个洁净无菌盖玻璃或玻璃底菜。把通道侧和紫外线治疗，直到乙醇蒸发。 </li><li>干燥后，翻转PDMS所以渠道都面临着向下延伸玻璃罩。按PDMS通道下放到玻璃板做一个良好的密封。添加无菌的PDMS覆盖的贴剂/关闭中心端口（端口B， 图2b）。允许继续之前彻底干燥。 </li><li>预涂层用无菌水10微克/毫升的胶原蛋白的信道的内侧。大衣，放置一个100微升滴在陈荫罴顶部埃尔，并用真空抽吸通过。后在37℃1小时，填充有胶原涂层溶液的冰箱传输通道。冷藏约15 - 30分钟。 </li><li>用吸气器或吸移管除去胶原涂布溶液，并开始胶原制备。 </li></ol> 5.胶原使用准备在微通道<ol><li>在冰上，中和胶原（1:1）用冰冷的100mM HEPES在2×PBS中，pH值7.4。彻底混合直到均匀（有关详细信息，请参见1.1节）。 </li><li>稀释中和胶原蛋白的适当浓度与细胞介质（有关详细信息，请参见1.2节）。孵育在冰上15分钟。 </li><li>同时，冷冻装在冰频道。 注意:我们的目标是对信道过程中的所有成分，在4℃或以下。胶原的成核温度和时间是到聚合过程中的关键参数，并且可以是用于进一步优化的起始点，如果需要的话。 </li><li> COUNT细胞与在此时间适当接种密度血球和重悬。为了便于计算，悬浮于1 - 3百万个细胞/毫升。 （更多详情，见第2.1节）。 </li><li>一旦细胞已被计算在内，15分钟过去了，进行胶原蛋白涌出。 </li></ol> 6.浇筑对齐和随机胶原蛋白微通道<ol><li>通过渠道绘图胶原蛋白之前，调整和设定真空压力内嵌真空调节器。真空压力提供了诱导和控制胶原的流速，它决定对准程度的力。 <ol><li>随机或不对齐的矩阵，可以使用一个宽的通道（3毫米宽×200微米高）用10毫米汞柱或更低的真空压力。 </li><li>对于排列矩阵，使用60毫米汞柱或更高的真空压力狭窄的通道（宽1mm×200微米高）。 </li></ol></li><li>从冰中取出安装通道和放置在层流H型清洁，消毒面洪水。 </li><li>迅速开展工作，并装载120 - 150微升中和胶原蛋白的成A口（ 图2b）。 </li><li>通过将25毫升移液管连接到真空管线以上端c（ 图2b）绘制通过通道胶原。通过在一个单一的，统一运动画胶原。重要信息:对于随机或对齐矩阵，得出胶原慢慢地穿过通道（约0.5 - 每秒1毫米），并停止一旦它到达终点。对于排列矩阵，得出整个胶原蛋白更快速，但是要注意避免产生气泡。 </li><li>小心地从P200的Pipetman或吸气港口区域去除多余的胶原蛋白。 </li><li>将无菌PDMS补丁在两个端口A和C.所有端口现在应该覆盖。 </li><li>经过2 - 3分钟，取出中心PDMS补丁（B口），并添加2 - 3微升细胞（5 - - 10个细胞）进入中心端口。允许部分聚合（转不透明）在室温下再10 - 15分钟。 </li><li> 10后- 15分钟后，转移至37℃另外15 - 30分钟以完成聚合。除去PDMS盖和介质添加根据需要完全覆盖通道和培养的细胞。细胞可以通过除去一个毫升旧媒体的，并用一个毫升新媒体代替它送入。 </li></ol>

<ol>
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3D胶原凝胶是一种宝贵的除了我们的工具箱，了解细胞如何解释和自己的局部微环境作出反应。该原稿提供了一个非常基本的协议用于3D胶原基质中嵌入的细胞，并重复地生成具有随机的或排列的胶原纤维基质。两个协议工作作为适应的平台，其中不同的胶原蛋白同种型，交联剂，或其它基质蛋白可能在聚合时加入。这也很容易修改的平台，以评估基因表达和蛋白生产不同的时段和端点。成像，评估蛋白质和RNA水平，以及阅读代谢特征都与这些3D技术非常兼容。 如同所有的技术很重要，重要的是评估凝胶和测定的质量是很重要的。质量好的胶原凝胶的标志是一个可以处理手动操作。例如，一个1毫升的[1毫克/毫升]胶原凝胶倒入一个六孔板应该很容易拿起一个镊子和操纵。如果凝胶眼泪或片段，凝胶的质量是次优的，和步骤需要采取提高胶原聚合。温度和pH值是良好的纤维形成和凝胶化所需的最重要的参数。首先确保所有组件都保持在冰上直至准备发生胶凝故障排除过程。这也是非常重要的，以确保中和缓冲液和胶原原液被充分混合。接下来，验证中和缓冲液的pH为pH 7.4。一个HEPES缓冲建议的PBS，因为它是对组织培养非常相容，具有广泛的缓冲范围，而储备溶液倾向于在4℃非常稳定长时间。它也有产生更长，更弯曲纤维，更类似于生理状态15的额外的好处。氢氧化钠是另一种的commo唯一一句用过中和溶液，但需要更紧密地滴定。这似乎也具有产生更短，更厚的纤维15的效果。无论如何，中和胶原的pH应当在pH 7.4。如果正在满足所有这些条件，以及凝胶的完整性仍不理想，那么问题在于股票胶原溶液。虽然库存胶原溶液具有在4℃6个月上市的保质期，我们已经发现，这是用于3D凝胶时比商业来源如权利要求一般较短。摒弃旧的瓶子，并从一个新的订单很多。这是不寻常的胶原蛋白从制造商的质量显著有所不同批次。 除了确定质量胶原凝胶，它也很重要，以确定是用于PDMS微通道内产生良好对准的关键步骤。这种技术的基本原理是，COLL的速率阿让流过所述微通道随后诱导长丝取向。因此，在控制纤维取向的程度最关键的步骤是在真空压力的适当的调制。一种用于在微通道随机胶原对准，真空压力必须低，并且胶原必须慢慢流下的通道。如果胶原流经过快，纤维将开始对齐。更宽的通道，使这一主张更容易。相反，对于好的胶原对准，必须使用更高的真空压力和胶原溶液必须通过更迅速地被拉出。在对准矩阵的生成的第二关键步骤是温度。以确保所有组件和解决方案是在使用前冰冷却是很重要的。如果频道没有被使用之前冷却，在通道中的胶原蛋白会聚合太快，并会导致更薄，更小的纤维。如果中和胶原在4℃冷却，而不是在冰中，enhanc编成核会引起较大的纤维直径而更难以由流动对齐。同样重要的是，以冷却所述微通道本身。没有事先心寒，胶原蛋白的小体积会热情很快产生效果差。 胶原微通道是一个非常有用的技术，但也有一定的局限性。为一体，在一个流动引起的微通道中的取向是从不完美的，绝不等于一个应变诱导取向的技术。总会有一些垂直于流动的方向取向的纤维，但可再现性，适应性和易用性大于小变化在对准通道内。更重要的是，细胞已被证明能有效检测在对准的微小变化，并且在这些通道3,15更持久的迁移反应。该细胞也将提高信道的取向为它们的数量增加，并且他们开始迁移。如果更大对准我s ^需要，PDMS微通道可以很容易地修改，以更窄的尺寸或有小的收缩，以提高对齐。两者都被证明是有效的，但具有其它相关的权衡16。另一个限制到微通道技术是与PDMS通道具有小的渗透性药物或荧光染料。这意味着这些化合物需要扩散向下从端口，这是令人惊讶的缓慢的信道，并且可以构成在洗出处理的困难。 虽然有一定的限制，以这个微通道的平台，它确实提供优于现有技术的几个优点。它是更实验获得，因为它不需要笨重仪器3,13或细胞用于产生对准的小种群。它也不需要外生磁珠13,14这往往是自体荧光和可能人为地交联的胶原蛋白网络。胶原微通道也简单地更具成本效益和经济。除了当前的优点，它也有可能在将来的应用中利用许多好处。为一体，在胶原微通道可设计或在几乎任何配置或实验修改。先进的设计允许信道来创建中空胶原管或三维结构，以模仿简化器官或肿瘤22的体系结构。此外，该信道可以由比PDMS等材料。这种潜在可能传达不同的机械性能，以嵌入胶原网络或基质。最后，用于该测定的试剂的量小也可能使较小的高通量筛选吸引力和经济。

Many cellular processes have been extensively studied in 2 dimensions, thereby forming a collective knowledge of basic cell signaling mechanisms. These studies, however, neglect important cellular cues received from the structural and mechanical properties of the local cellular microenvironment and extracellular matrix (ECM). To better understand how cells interact within a physiological context, it is important to study them in 3-dimensional (3D) assays. The ECM for these 3D assays can either be cell-derived or reconstituted from purified proteins. Regardless of the source of the ECM, 3D matrix assays have proven to be invaluable for understanding how cells navigate and interact within the physiological world. For example, cells grown in 3D matrices display distinct modes of locomotion that depend on the mechanical nature of their surrounding ECM which are not observed in 2D experiments4-6. Moreover, cells cultured in 3D also have fewer and less pronounced stress fibers and focal adhesions than their counterparts grown on hard surfaces such as glass or plastic7. 
The importance of contextual 3D assays is not limited to cell migration, however. Some other cell signaling events can only be investigated through the use of 3D assays. During tissue and cell differentiation, the stiffness of the extracellular environment and ECM provides signals that can influence morphogenic events. For example, mammary epithelial tubulogenesis only occurs in low stiffness 3D matrices, but not in stiff matrices nor on 2D substrata8,9. When cultured within stiff 3D matrices, these same epithelial cells take on an aberrant phenotype with increased proliferation and cell membrane protrusions driven through altered FAK and ERK signaling10. Many other signaling pathways and cellular processes are known to be similarly affected by the stiffness of the local cellular environment, and these signaling cascades highlight the importance of investigating signaling events and cellular phenotype in the context of appropriate local mechanical properties of a 3D ECM. 
Collagen I is a particularly relevant protein to use for in vitro studies as it is the most abundant component of the ECM and is responsible for many of the mechanical properties of the cellular microenvironment. While it was originally thought of as merely a structural protein, its role is now known to be much more complex. Collagen fiber composition, architecture, orientation, density, and stiffness all provide a concentrated milieu of signaling information5. During the progression of certain diseases, such as chronic inflammation and tumorigenesis, the collagen network is dramatically remodeled2,11. More specifically in breast cancers, increased collagen deposition and tissue stiffness accompany and likely contribute to tumor progression. In these early tumors, the stiffened collagen network appears strained and more aligned, such that most of the fibers encapsulate the growing tumor2. As the tumor progresses, the collagen continues to reorganize, and regions of the fibrillar network become orientated perpendicular to the tumor boundary2,12. Perpendicular alignment serves as a prognostic biomarker where these patients have a poorer disease free progression and overall survival1. One explanation for this correlation is that the poor outcomes are a consequence of increased dissemination of tumor cells via persistent cell migration in aligned collagen networks3. 
To understand how cells specifically respond to alignment and organization that is observed in tumor progression, it is necessary to generate both random and aligned 3D collagen matrices for experimentation. There are three basic methodologies to induce alignment within fibrillar networks. The first technique utilizes a strain-inducing device where the collagen between two points is contracted or stretched to generate alignment. Fibers parallel to the axis of force are pulled taut while fibers perpendicular to the axis are compressed and buckled. While strain-induced techniques typically offer superb alignment, this approach requires bulky equipment that is not easily adaptable to many platforms3,13. Alternatively, cell-induced strain can be created by placing localized plugs of cells that subsequently contract and align the collagen13. This method has the problem of being variable, as many parameters may be subject to change. The second method utilizes magnetic beads and a magnetic field during polymerization to induce collagen alignment13,14. Good results can be obtained from this method with unsophisticated equipment, but it does require the use of antibodies or some other method to magnetize the polymer. Therefore, it can be somewhat expensive to use, and the stiffness of the collagen gel is potentially modified by the increased connections in the network. Moreover, the magnetic beads used in this process are often autofluorescent, which is problematic for imaging experiments. Lastly, alignment can be generated by PDMS microfluidic channels3,15,16. In this method, collagen alignment is achieved by flowing polymerizing collagen through small microfluidic channels. These microfluidic channels can be made in a multitude of designs, and are easily adaptable to many platforms. Moreover, they are very economical as very small quantities of collagen and other reagents are used due to their diminutive sizes.
Provided here is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. In addition, a more advanced technique, wherein PDMS microfluidic channels are used to control the organization and alignment of the collagen matrix is also provided. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a 3-dimensional, physiological microenvironment.

<table><tbody><tr><td>High Concentration Rat Tail Collagen</td><td>Corning</td><td>354249</td><td></td></tr><tr><td>SylGard184 elastomer kit</td><td>Corning</td><td>NC9285739</td><td>Elastomer for PDMS channels</td></tr><tr><td>HEPES</td><td>Fisher</td><td>BP310</td><td>For HEPES neutralization buffer</td></tr><tr><td>KCl&amp;nbsp;</td><td>Fisher</td><td>BP366</td><td>For HEPES neutralization buffer</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher</td><td>BP362</td><td>For HEPES neutralization buffer</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher</td><td>S374</td><td>For HEPES neutralization buffer</td></tr><tr><td>NaCl</td><td>Fisher</td><td>BP358</td><td>For HEPES neutralization buffer</td></tr><tr><td>Levy Improved Neubauer Hemacytometer</td><td>Fisher</td><td>15170-208</td><td>cell counting</td></tr><tr><td>6-well non-tissue culture plate&amp;nbsp;</td><td>Corning</td><td>351146</td><td></td></tr><tr><td>50 mm glass bottom dish</td><td>MatTek</td><td>P50g-1.5-30-f</td><td></td></tr><tr><td>Bel-Art Plastic Vacuum Desiccator</td><td>Bel-Art</td><td>F4200-2021</td><td>Degassing chamber for PDMS</td></tr><tr><td>transparency film&amp;nbsp;</td><td>3M</td><td>pp2950</td><td>Plastic film for pouring pdms channels</td></tr><tr><td>ThermoScientific CimaRec</td><td>ThermoScientific&amp;nbsp;</td><td>HP141925</td><td>Hot plate for curing PDMS microchannels</td></tr><tr><td>Vacuum regulator</td><td>Precision Medical</td><td>PM3100</td><td>Vacuum regulator for collagen microchannels</td></tr><tr><td>8&quot; x 8&quot; rubber sheet</td><td>Amazon - Rubber-Cal</td><td>Silicone - 60A</td><td>rubber sheet for pouring PDMS microchannel</td></tr><tr><td>8&quot; x 8&quot; x 0.125&quot; acrylic sheet</td><td>Amazon</td><td>Plexiglass sheets</td><td>for pouring PDMS microchannels</td></tr><tr><td>10 lb weights</td><td>Amazon</td><td>CAP Barbell</td><td>for pouring PDMS microchannels</td></tr><tr><td>15 ml Conical tubes</td><td>Fisher</td><td>352097</td><td></td></tr><tr><td>50 ml Conical tubes</td><td>Fisher</td><td>352098</td><td></td></tr><tr><td>Plastic pipets</td><td>Dot Scientific</td><td>229202B, 229206B, and 667225B</td><td>2&amp;nbsp;ml, 5&amp;nbsp;ml, and 25&amp;nbsp;ml</td></tr><tr><td>70% EtOH</td><td>Fisher</td><td>NC9663244</td><td></td></tr></tbody></table>

preparation of 3d collagen gels and microchannels for the study of 3d interactions in vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

而三维测定可以胶原凝胶的相同的刚度中完成，改变凝胶硬度可以用来确定细胞将如何在它们的细胞微环境的机械变化。僵硬胶原凝胶被定义为凝胶，其中嵌入的细胞不能在本地收缩周边的胶原蛋白。不同细胞类型的固有收缩是唯一的，并且因此最好是开始用一个简单的收缩曲线建立将被感测为标准，僵硬的胶原浓度。 通过保持细胞数量恒定，增加胶原蛋白凝胶的浓度开始。凝胶的刚性将与浓度呈指数扩展，如前面10,20所示。一个收缩测定的代表性例子示于图1。在这里，5 4T1细胞嵌入eithe岭2毫克/毫升或5天3.5毫克/毫升的胶原凝胶。的4T1细胞系是高度收缩，转移性癌症线，但仅能够由大约10％至收缩高密度凝胶（ 图1a中的C）。这种低收缩的水平是典型的刚性凝胶分类。可比低密度凝胶（2毫克/毫升， 图1b，c）的同一时间帧期间收缩57％（N = 4）。要注意的是增加胶原配体的量可影响细胞信号传导途径，因此第二装置来改变刚度保持胶原浓度恒定并通过被留下附着于底部比较被抑制凝胶（硬）是非常重要的菜或者是因为他们已经发布在细胞培养基中自由浮动收缩（标准）。这可以作为一个良好的控制，以确定在细胞信号的变化是否从增加的胶原配位体或基体的硬度的结果。 <p class="jove_content" fo:keep-together.within页="1"&gt;虽然胶原凝胶收缩试验是直截了当的，也有一些需要注意的事项使用这些类型的测定时的感觉。为一体，高密度的条件下，以验证不存在细胞活力的损失是很重要的。我们已经观察到有该细胞会容忍微环境的刚度天花板，并已观察到增加的凋亡或细胞死亡在某些细胞系，这会导致随后的结果被误解。应该有细胞生存力的高，低刚度条件之间没有明显的损失。这可以通过Western印迹对胱天蛋白酶活性或一些其它测定用于测定细胞生存力来确定。其次，胶原凝胶也可收缩太多从而创造一个更硬的环境，并减少高和低刚度状况之间的差异。我们建议不超过60收缩 - 70％。为了最大限度地提高重复性，以优化时间是非常重要的实验中，初始细胞接种密度，以及矩阵所使用的细胞类型的收缩刚度帧。 一旦三维基质内包埋细胞的掌握实现，测定可以进行修改，以改变该胶原纤维的组织和取向。这是通过使用PDMS微流体通道（ 图2）实现的。对于本手稿的目的，两个类似的构象被使用，窄和宽通道，带3的开口或端口（ 图2b）。在典型的实验，以确定相对于对准矩阵随机的细胞反应，未聚合的胶原是通过微流体通道绘制从端口A到端口C的与被添加到端口B细胞胶原取向的程度是通过胶原的速率调制流，使得较高的胶原蛋白的流速产生更好的调整。使用较窄的通道加上较高真空压力增强了胶原网络的取向，同时配合使用与低真空压力下更宽的通道产生随机矩阵。纤维状网络的组织可以通过二次谐波产生可视化，如图代表性图像（ 图2c，d）所示 。在这些图像中，胶原纤维（由箭头表示，并且在紫色伪彩色）与在窄通道（ 图2d）胶原流的轴线对齐更一致。在宽的信道的胶原纤维具有多种取向，并具有更多的随机分布（ 图2c）。 宽和窄的通道之间的差异是可以辨认的人的眼睛，但也可以通过专门为纤维分析开发的软件进行分析。 CT-火灾，开源和免费的软件，采用了预处理其次是基于曲波算法纤维提取算法量化纤维角度，厚度，长度和直线21。对于每个条件，超过500张图像，随机从通道内的不同位置取样，提取并分析超过125000纤维经CT-FIRE。为狭窄的通道（ 图2F）中产生的随后的纤维角度柱状图示出平行于流动的方向取向的纤维的清晰富集。与此相反，宽信道具有更多的等效纤维角度分布（ 图2e），这是一个更随机矩阵一致。 重要的是，使用微流体装置的，作为一种技术，以控制组织和胶原纤维的取向，允许对改变的矩阵条件的各种细胞应答的评估。例如，宋等人 。确定人类乳腺fibrobasts的可行性取决于胶原纤维。15此外，最近从我们的实验室工作表明，乳腺癌细胞相比随机胶原基质更持久地迁移对齐的胶原纤维。3这些结果有助于更好地确定微环境的乳腺生物学和乳腺癌的范围内的作用。向前移动，以精确地控制在3D细胞微环境将允许许多不同的疾病状态的范围内的重要的细胞响应继续调查的能力。 <img alt="图1" src="/files/ftp_upload/53989/53989fig1.jpg" /> 图1. 3D胶原凝胶为细胞收缩和细胞反应测量到ECM刚度。在这两个接种4T1细胞 （ 一 ）3.5毫克/毫升和 （b）第 2毫克/毫升胶原凝胶并使其生长5天。随着时间的推移，胶原凝胶（灰色区虚线内）将收缩，直径测量和量化（C，学生t检验，误差棒+/- SD）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53989/53989fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53989/53989fig2.jpg" /> 图2.产生随机和对准胶原基质使用的PDMS微通道。一个六英寸硅晶片（ 一 ）作为两个宽和窄的PDMS微通道（b）一种模板。相同胶原浓度通过这两种类型的信道的拉伸，和单个纤维（箭头，C，D）与流动方向对齐（箭头）中窄微通道 （ 四 ）。微通道宽更产生随机矩阵。光纤ANGL相对于图象的底部，ES进行计数，（e）中和（f）示出。比例尺= 26微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53989/53989fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo at JoVE.com

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

胶原是细胞外基质的核心组件，并提供必要的暗示了几个细胞过程，从移植到分化和增殖。这里提供了一种用于三维胶原凝胶内嵌入电池的协议，和用于产生使用的PDMS微通道的随机或对齐胶原基质的更先进的技术。

3D胶原凝胶和微通道3D相互作用的研究准备<em&gt;在体内</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

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Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

16.9K 次观看.  University of Wisconsin-Madison. 胶原是细胞外基质的核心组件，并提供必要的暗示了几个细胞过程，从移植到分化和增殖。这里提供了一种用于三维胶原凝胶内嵌入电池的协议，和用于产生使用的PDMS微通道的随机或对齐胶原基质的更先进的技术。 

视频: Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized tissues.  The modules can be made of collagen as well as other gelable or crosslinkable materials.  They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells.  The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable.  These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues.  There are three main steps to making the modules: (1) neutralizing the collagen and embedding cells in it, (2) gelling the collagen in the tube and cutting the modules and (3) coating the modules with endothelial cells.

Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.

微组织使用胶原蛋白的模块的制备凝胶含有细胞

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

科研

生物工程

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

器官Ⅰ型胶原的含量可延展的平台，以评估细胞行为的一个3维的情况下

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Revealing the Cytoskeletal Organization of Invasive Cancer Cells in 3D

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration in more appropriate 3D environments, which better resemble the dimensionality of the physiological processes in question. Migratory cells can substantially differ in their morphology and mode of migration depending on whether they are moving on 2D or 3D substrates. Due to technical difficulties and incompatibilities with most standard protocols, structural and functional analysis of cells embedded within 3D matrices still remains uncommon. This article describes methods for preparation and imaging of 3D cancer cell cultures, either as single cells or spheroids. As an appropriate ECM substrate for cancer cell migration, we use nonpepsinized rat tail collagen I polymerized at room-temperature and fluorescently labeled to facilitate visualization using standard confocal microscopes. This work also includes a protocol for 3D immunofluorescent labeling of endogenous cell cytoskeleton. Using these protocols we hope to contribute to a better description of the molecular composition, localization, and functions of cellular structures in 3D.

This article presents a method to fluorescently label collagen that can be further used for both fix and live imaging of 3D cell cultures. We also provide an optimized protocol to visualize endogenous cytoskeletal proteins of cells cultured in 3D environments.

在3D揭示侵入性癌症细胞骨架组织

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration ...

医学

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to tumor and cancer metastasis. Despite intense research efforts, the basic biochemical and biophysical principles of cell migration are still not fully understood, especially in the physiologically relevant three-dimensional (3D) microenvironments. Here, we describe an in vitro assay designed to allow quantitative examination of 3D cell migration behaviors. The method exploits the cell&#8217;s mechanosensing ability and propensity to migrate into previously unoccupied extracellular matrix (ECM). We use the invasion of highly invasive breast cancer cells, MDA-MB-231, in collagen gels as a model system. The spread of cell population and the migration dynamics of individual cells over weeks of culture can be monitored using live-cell imaging and analyzed to extract spatiotemporally-resolved data. Furthermore, the method is easily adaptable for diverse extracellular matrices, thus offering a simple yet powerful way to investigate the role of biophysical factors in the microenvironment on cell migration.

The mechanical properties and microstructure of the extracellular matrix strongly affect 3D migration of cells. An in vitro method to study the spatiotemporal cell migration behavior in biophysically variable environments, at both population and individual cell levels, is described.

同心凝胶系统来研究基质微环境的三维细胞迁移的生物物理作用

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

工程三维细胞化胶原凝胶的血管组织再生

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

平面梯度扩散系统的调查趋化三维胶原基质

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

层与层胶原沉积在微流体装置的Microtissue稳定

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

缩微和3D微血管大会

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Microengineering 3D Collagen Hydrogels with Long-Range Fiber Alignment

Aligned collagen I (COL1) fibers guide tumor cell motility, influence endothelial cell morphology, control stem cell differentiation, and are a hallmark of cardiac and musculoskeletal tissues. To study cell response to aligned microenvironments in vitro, several protocols have been developed to generate COL1 matrices with defined fiber alignment, including magnetic, mechanical, cell-based, and microfluidic methods. Of these, microfluidic approaches offer advanced capabilities such as accurate control over fluid flows and the cellular microenvironment. However, the microfluidic approaches to generate aligned COL1 matrices for advanced in vitro culture platforms have been limited to thin "mats" (&lt;40 &#181;m in thickness) of COL1 fibers that extend over distances less than 500 &#181;m and are not conducive to 3D cell culture applications. Here, we present a protocol to fabricate 3D COL1 matrices (130-250 &#181;m in thickness) with millimeter-scale regions of defined fiber alignment in a microfluidic device. This platform provides advanced cell culture capabilities to model structured tissue microenvironments by providing direct access to the micro-engineered matrix for cell culture.

This protocol demonstrates the use of a microfluidic channel with changing geometry along the fluid flow direction to generate extensional strain (stretching) to align fibers in a 3D collagen hydrogel (&lt;250 &#181;m in thickness). The resulting alignment extends across several millimeters and is influenced by the extensional strain rate.

具有长距离纤维排列的微工程3D胶原蛋白水凝胶

Aligned collagen I (COL1) fibers guide tumor cell motility, influence endothelial cell morphology, control stem cell differentiation, and are a hallmark ...

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is centered on culturing small pieces of embryonic or early postnatal rodent brains inside a 3-D hydrogel formed by the rat tail tendon-derived type I collagen with specific porosity. Tissue pieces are cultured inside the hydrogel and confronted to specific brain fragments or genetically-modified cell aggregates to produce and secrete molecules suitable for creating a gradient inside the porous matrix. The steps of this protocol are simple and reproducible but include critical steps to be considered carefully during its development. Moreover, the behavior of growing axons can be monitored and analyzed directly using a phase-contrast microscope or mono/multiphoton fluorescence microscope after fixation by immunocytochemical methods.

Here, we provide a method for analyzing the behavior of growing axons in 3D matrices, mimicking their natural development.

生成基于3-D胶原蛋白的水凝胶,分析神经系统发育过程中的Axonal生长和行为

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...

3D胶原凝胶和微通道3D相互作用的研究准备

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